Global regulation by proteolysis in Escherichia coli: Molecular recognition, signal integration and quantitative analysis of proteolysis-controlled regulatory circuits

某些关键调节因子的细胞数量是合成和降解调节之间高度动态和调节平衡的结果。以大肠杆菌中RNA聚合酶Sigma-S亚基的多重应激抑制蛋白水解为模型系统，本文提出的建议侧重于调节蛋白水解领域的两个主要未解决的问题：(i)蛋白水解机制对选择性底物识别的分子细节，以及（ii）将环境应激条件与关键细胞调节因子的蛋白水解速率联系起来的信号转导途径。Sigma-S被识别因子RssB直接靶向，RssB对Sigma-S内一个小区域（a2.5）的亲和力由磷酸化控制。然后RssB将Sigma-S转移到ClpXP蛋白酶，在那里Sigma-S被迅速完全降解。将研究Sigma-S蛋白水解识别位点（可能也包括Sigma-70）与各种识别因子和伴侣（RssB、ClpX、ClpA）之间的分子相互作用。此外，还将阐明在各种应激条件下抑制Sigma-S转换的信号转导途径。这些途径分别控制RssB磷酸化和/或与Sigma-S和ClpXP的竞争性结合伙伴（如核心RNA聚合酶或替代ClpXP底物）的细胞数量和相互作用。这些研究应该为调节蛋白水解以及复杂的细菌信号整合网络提供重要的新见解。

The cellular amount of certain key regulatory factors is the result of a highly dynamic and regulated balance between regulation of synthesis and degradation. Using the multiple stress-inhibited proteolysis of the Sigma-S subunit of RNA polymerase in E.coli as a model system, the proposal submitted here focusses on the two major unsolved problems in the field of regulated proteolysis: (i) the molecular details of selective substrate recognition by the proteolytic machinery, and (ii) the signal transduction pathways that connect environmental stress conditions to the rate of proteolysis of a key cellular regulator. Sigma-S is directly targeted by a recognition factor, RssB, whose affinity for a small region within Sigma-S (a2.5) is controlled by phosphorylation. RssB then transfers Sigma-S to the ClpXP protease, where Sigma-S is rapidly and completely degraded. Molecular interactions between proteolytic recognition sites in Sigma-S (and probably in Sigma-70) and various recognizing factors and chaperones (RssB, ClpX, ClpA) will be studied. In addition, the signal transduction pathways that inhibit Sigma-S turnover in response to various stress conditions will be elucidated. These pathways either control RssB phosphorylation and/or the cellular amount of and interaction with competing binding partners for Sigma-S and ClpXP, such as core RNA polymerase or alternative ClpXP substrates, respectively. These studies should provide important novel insights into regulated proteolysis as well as into complex bacterial signal integration networks in general.